CN114672560A

CN114672560A - Detection kit and method for identifying colorectal cancer state through exosome miRNA marker

Info

Publication number: CN114672560A
Application number: CN202210032516.9A
Authority: CN
Inventors: 胡鹏程; 李明明; 蒲珏
Original assignee: Beijing Exellon Medical Technology Co ltd
Current assignee: Beijing Exellon Medical Technology Co ltd
Priority date: 2022-01-12
Filing date: 2022-01-12
Publication date: 2022-06-28

Abstract

Provided herein is a test kit for use in the diagnosis of a colorectal cancer state in a subject comprising a detection reagent for detecting the level of a miRNA biomarker selected from the group consisting of hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and hsa-miR-33b-3p, and any combination thereof, in a biological sample from the subject. Also provided herein are methods of diagnosing a colorectal cancer status in a subject. The detection kit and the method provided by the invention provide a new efficient, noninvasive and accurate way for screening, diagnosing and prognosticating the early colorectal cancer.

Description

Detection kit and method for identifying colorectal cancer state through exosome miRNA marker

Technical Field

The invention belongs to the field of genetic engineering and oncology, and particularly relates to an exosome miRNA marker related to colorectal cancer auxiliary diagnosis and application thereof.

Background

Colorectal cancer is one of the main cancers threatening the life health of people in China and all over the world, and causes serious social burden. The latest '2020 global cancer report' issued by the international agency for research on cancer (IARC) under the world health organization shows 1929 ten thousand new cancer cases worldwide in 2020, of which 457 thousand new cancers in china account for 23.7% of the world. The global new number of colorectal cancers in 2020 is 193 ten thousand, second only to breast cancer and lung cancer. In China, the new number of colorectal cancers in 2020 is 56 ten thousand, and has become the second largest cancer species after lung cancer. With the increasing aging population, the 2040 year cancer burden is expected to increase by 50%, at which point the number of new cancer cases in the country will reach nearly 3000 tens of thousands, and this trend is most pronounced in countries experiencing social and economic changes. The occurrence and development of colorectal cancer mostly follow an adenoma-carcinoma sequence, the time for the colorectal cancer to progress from precancerous lesion to carcinoma is generally 5-10 years, and an important time window is provided for early diagnosis and clinical intervention of the disease. Furthermore, the prognosis of colorectal cancer is closely related to the diagnostic stage. The 5-year relative survival rate for stage i colorectal cancer is 90%, while the 5-year relative survival rate for stage iv colorectal cancer with distant metastasis is only 14%. Numerous studies and practices have shown that colorectal cancer screening and early diagnosis and treatment can effectively reduce the mortality rate of colorectal cancer.

Colonoscopy is a unique and irreplaceable position in colorectal cancer screening and is a core link of the whole colorectal cancer screening process, pathological examination of biopsy resection specimens under the colonoscope is a golden standard for colorectal cancer diagnosis, and the morbidity and mortality of colorectal cancer can be reduced by removing precancerous lesions under the colonoscope. The colonoscope can directly observe the inner wall of the colorectal cavity, which is the most sensitive method for finding intestinal tumors, but the colonoscopy still has a certain misdiagnosis rate, mainly occurs in the proximal colon, and mainly comprises jagged polyps and flat adenomas. Obtaining good bowel preparation, performing standardized colonoscopy procedures and fine, refractory endoscopic observations are important measures to reduce the rate of missed diagnoses of lesions, and therefore colonoscopy has high requirements on both the subject and the endoscopist. Due to dietary restrictions and strict bowel cleansing preparations prior to colonoscopy, some subjects who do not receive sedation or anesthesia colonoscopy experience significant pain, resulting in poor compliance. In addition, the direct and indirect costs of colonoscopy also affect the willingness of the population to participate in the screening; moreover, colonoscopy is invasive and has a certain complication rate, and target people often refuse colonoscopy due to fear. Research data at home and abroad show that even if the fecal occult blood test is positive, the proportion of subsequent colonoscopy is only 30-40%. CT, PET-CT, MRI, ultrasound and the like are all conventional detection methods for colorectal cancer in the aspect of imaging, compared with colonoscopes, the imaging examination of colorectal cancer has the advantages of non-invasiveness, convenience, rapidness and the like, and the detection compliance of a person to be detected is also higher, but the methods have strict requirements on preparation before examination of a patient and the technology of a doctor, and have the problems of false positive, radiation hazard and the like. Due to the limitations of imaging examination, it is difficult to meet the urgent need of large-scale screening, and with the research in the field of molecular biology, people begin to move their eyes to molecular markers for early diagnosis of colorectal cancer. The methylation detection of plasma Septin9 gene and the detection of fecal pyruvate kinase (M2-PK) are the currently widely applied molecular screening methods for colorectal cancer, and recently, a large-scale clinical test in China finds that the sensitivity and specificity of the methylation detection of plasma Septin9 gene for diagnosing colorectal cancer are respectively 74.8 percent and 87.4 percent, which are higher than those of FIT detection. However, mSEPT9 has insufficient diagnosis sensitivity and specificity for precancerous lesions (colorectal adenomas and polyps, advanced adenomas), is not recommended to be used for population screening, and can be used as a selection and supplement for individual diagnosis.

Exosomes (exosomes) are small vesicles of about 30-150nm in diameter secreted by living cells, widely distributed in various body fluids of humans. Exosomes derived from different tissues not only have their specific protein molecules, but also contain key molecules for their function. Exosomes are secreted and released by cells, spread in body fluids such as blood and then phagocytosed by other cells, and are important mediators of intercellular communication. Research shows that exosome plays an important role in the processes of tumorigenesis, invasion, metastasis and the like.

mirnas are an endogenous class of non-coding RNAs found in eukaryotes with post-transcriptional regulatory functions, typically 19-25 nucleotides in length. As a class of small, non-coding single stranded RNAs, they can silence target mrnas by binding to the corresponding 3 '-untranslated region (3' -UTR) or open reading frame. Numerous studies have shown that mirnas are involved in the regulation of a variety of physiological processes including immune defense, immune surveillance, immune homeostasis, and the development of tumorigenesis, and thus many circulating mirnas have been used for the diagnosis, prognosis, and treatment of tumors. In 2008, an American researcher reports that circulating miRNA has potential to become a novel marker of solid tumor for the first time, and subsequent researches evaluate the feasibility of miR-21 and miR-92a in a fecal sample in the aspect of distinguishing colorectal cancer from colorectal polyps, and find that miR-21 and miR-92a have good diagnostic capability on colorectal cancer and colorectal polyps. However, circulating free mirnas under physiological conditions have poor stability, limiting their application as reliable biomarkers. With the progress of research, researchers find that miRNA in extracellular vesicles has strong consistency with a precursor before secretion, and due to the lipid bilayer membrane structure of the vesicles (exosomes), the content of the miRNA has strong stability, and the application of the miRNA in various application fields is greatly improved.

In the face of the increasingly severe colorectal cancer occurrence and development situation, how to effectively reduce the burden of colorectal cancer diseases in China becomes a major public health problem to be urgently solved.

Disclosure of Invention

In one aspect, provided herein is a test kit for diagnosing a colorectal cancer status in a subject, comprising a detection reagent for detecting the level of a miRNA biomarker selected from the group consisting of hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and hsa-miR-33b-3p, and any combination thereof, in a biological sample from the subject.

In some embodiments, the miRNA biomarker comprises hsa-miR-320a-3p, hsa-miR-19b-3p, or hsa-miR-196a-5 p; comprises hsa-miR-320a-3p, hsa-miR-31-5p and hsa-miR-409-5 p; or comprises hsa-miR-19b-3p, hsa-miR-196a-5p and hsa-miR-33b-3 p.

In some embodiments, the miRNA biomarkers are hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and hsa-miR-33b-3 p.

In some embodiments, the colorectal cancer status comprises a colorectal cancer susceptibility or a presence, progression, subtype, stage and/or differentiation status of colorectal cancer.

In some embodiments, the colorectal cancer status is stage I or stage II of colorectal cancer, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19b-3p, and/or hsa-miR-196a-5 p.

In some embodiments, the colorectal cancer status is adenocarcinoma and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19a-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, and/or hsa-miR-33b-3 p.

In some embodiments, the colorectal cancer status is mucinous adenocarcinoma and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-31-5p, hsa-miR-196a-5p, and/or hsa-miR-19b-3 p.

In some embodiments, the colorectal cancer status is signet ring cell carcinoma and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-92a-3p, hsa-miR-196a-5p, and/or hsa-miR-33b-3 p.

In some embodiments, the colorectal cancer status is other types of colorectal cancer and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and/or hsa-miR-33b-3 p.

In some embodiments, the detection reagent that detects miRNA biomarker levels comprises a reverse transcription primer, a PCR amplification primer pair, and/or a Taqman probe.

In some embodiments, the detection reagents for detecting the level of hsa-miR-320a-3p include: comprising SEQ ID NO: 10, and a reverse transcription primer; comprising SEQ ID NO: 19 and 28; and/or comprises SEQ ID NO: 29, and a Taqman probe; the detection reagent for detecting the level of hsa-miR-19a-3p comprises: comprises the amino acid sequence of SEQ ID NO: 11, and a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 20 and 28; and/or comprises SEQ ID NO: 30, a Taqman probe; the detection reagent for detecting the level of hsa-miR-19b-3p comprises: comprises the amino acid sequence of SEQ ID NO: 12; comprises the amino acid sequence of SEQ ID NO: 21 and 28; and/or comprises SEQ ID NO: 31, a Taqman probe; the detection reagent for detecting the level of hsa-miR-31-5p comprises: comprises the amino acid sequence of SEQ ID NO: 13, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 22 and 28; and/or comprises SEQ ID NO: 32, a Taqman probe; the detection reagent for detecting the level of hsa-miR-92a-3p comprises: comprises the amino acid sequence of SEQ ID NO: 14, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 23 and 28; and/or comprises SEQ ID NO: 33, a Taqman probe; the detection reagent for detecting the level of hsa-miR-196a-5p comprises: comprises the amino acid sequence of SEQ ID NO: 15, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 24 and 28; and/or comprises SEQ ID NO: 34, 34 is a Taqman probe; the detection reagent for detecting the level of hsa-miR-409-5p comprises: comprises the amino acid sequence of SEQ ID NO: 16; comprises the amino acid sequence of SEQ ID NO: 25 and 28; and/or comprises SEQ ID NO: 35 in sequence as shown in the specification; the detection reagent for detecting the level of hsa-miR-520d-5p comprises: comprises the amino acid sequence of SEQ ID NO: 17; comprises the amino acid sequence of SEQ ID NO: 26 and 28; and/or comprises SEQ ID NO: 36, a Taqman probe; and the detection reagent for detecting the level of hsa-miR-33b-3p comprises: comprises the amino acid sequence of SEQ ID NO: 18, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 27 and 28; and/or comprises SEQ ID NO: 37, and a Taqman probe having the sequence shown in the specification.

In some embodiments, the test kit further comprises a detection reagent for detecting the level of hsa-miR-let-7d as an internal reference gene; the detection reagent comprises: comprising SEQ ID NO: 38; comprises the amino acid sequence of SEQ ID NO: 39 and 28; and/or comprises SEQ ID NO: 40 in sequence shown in the specification.

In some embodiments, the test kit further comprises reagents for isolating exosomes from the biological sample; optionally, further comprising reagents for extracting miRNA from the exosomes.

In some embodiments, the test kit further comprises instructions for determining colorectal cancer status in the subject based on logistic regression based on the miRNA biomarker levels.

In some embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, stool, milk, ascites, urine, and tissue biopsy; preferably plasma, serum or feces.

In another aspect, provided herein is a method of identifying colorectal cancer status in a subject, comprising: 1) isolating exosomes from a biological sample from the subject; 2) detecting a level of a miRNA biomarker in the exosomes, the miRNA biomarker selected from the group consisting of hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p and hsa-miR-33b-3p and any combination thereof; and 3) comparing the miRNA biomarker levels detected in step 2) with the levels of corresponding miRNAs in a population to determine the colorectal cancer status in the subject.

In some embodiments, the miRNA biomarkers include a miRNA biomarker comprising hsa-miR-320a-3p, hsa-miR-19b-3p, or hsa-miR-196a-5 p; comprises hsa-miR-320a-3p, hsa-miR-31-5p and hsa-miR-409-5 p; or comprises hsa-miR-19b-3p, hsa-miR-196a-5p and hsa-miR-33b-3 p.

In some embodiments, the detecting of the miRNA biomarker levels in step 2) comprises using miRNA biomarker-specific detection reagents comprising reverse transcription primers, PCR amplification primer pairs, and/or Taqman probes.

In some embodiments, the detection reagents for detecting the level of hsa-miR-320a-3p include: comprising SEQ ID NO: 10, and a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 19 and 28; and/or comprises SEQ ID NO: 29, and a Taqman probe; the detection reagent for detecting the level of hsa-miR-19a-3p comprises: comprises the amino acid sequence of SEQ ID NO: 11, and a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 20 and 28; and/or comprises SEQ ID NO: 30, a Taqman probe; the detection reagent for detecting the level of hsa-miR-19b-3p comprises: comprises the amino acid sequence of SEQ ID NO: 12; comprises the amino acid sequence of SEQ ID NO: 21 and 28; and/or comprises SEQ ID NO: 31, a Taqman probe; the detection reagent for detecting the level of hsa-miR-31-5p comprises: comprises the amino acid sequence of SEQ ID NO: 13, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 22 and 28; and/or comprises SEQ ID NO: 32, a Taqman probe; the detection reagent for detecting the level of hsa-miR-92a-3p comprises: comprises the amino acid sequence of SEQ ID NO: 14, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 23 and 28; and/or comprises SEQ ID NO: 33, a Taqman probe; the detection reagent for detecting the level of hsa-miR-196a-5p comprises: comprises the amino acid sequence of SEQ ID NO: 15, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 24 and 28; and/or comprises SEQ ID NO: 34, 34 is a Taqman probe; the detection reagent for detecting the level of hsa-miR-409-5p comprises: comprises the amino acid sequence of SEQ ID NO: 16; comprises the amino acid sequence of SEQ ID NO: 25 and 28; and/or comprises SEQ ID NO: 35, a Taqman probe with a sequence shown in the specification; the detection reagent for detecting the level of hsa-miR-520d-5p comprises: comprises the amino acid sequence of SEQ ID NO: 17; comprises the amino acid sequence of SEQ ID NO: 26 and 28; and/or comprises SEQ ID NO: 36, a Taqman probe; and the detection reagent for detecting the level of hsa-miR-33b-3p comprises: comprises the amino acid sequence of SEQ ID NO: 18, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 27 and 28; and/or comprises SEQ ID NO: 37, and a Taqman probe having the sequence shown in the specification.

In some embodiments, step 2) further comprises detecting the level of hsa-miR-let-7d as an internal reference gene; the detection reagent for detecting the level of hsa-miR-let-7d comprises: comprising SEQ ID NO: 38; comprises the amino acid sequence of SEQ ID NO: 39 and 28; and/or comprises SEQ ID NO: 40 in sequence shown in the specification.

In some embodiments, step 3) comprises determining colorectal cancer status in the subject based on logistic regression according to the miRNA biomarker levels.

The detection kit and the method provided by the invention can provide a new efficient, noninvasive and accurate approach for screening, diagnosing and prognosticating early colorectal cancer.

Drawings

Figure 1 shows the horizontal distribution (relative expression) of 9 exosome miRNA biomarkers in different colorectal cancer stages. ***: the P value is less than or equal to 0.001; **: p is more than 0.001 and less than or equal to 0.01; *: p value is more than 0.01 and less than or equal to 0.05; ns: p value > 0.05.

Figure 2 shows the horizontal distribution (relative expression) of 9 exosome miRNA biomarkers in different colorectal cancer subtypes. ***: the P value is less than or equal to 0.001; **: p is more than 0.001 and less than or equal to 0.01; *: p value is more than 0.01 and less than or equal to 0.05; ns: p value > 0.05.

Figure 3 shows Receiver Operating Characteristic (ROC) curves for single miRNA markers and different miRNA marker combinations for colorectal cancer diagnosis. (A) hsa-miR-320a-3p alone; (B) hsa-miR-19b-3p alone; (C) hsa-miR-196a-5p alone; (D) combining hsa-miR-320a-3p + hsa-miR-31-5p + hsa-miR-409-5 p; (E) combining hsa-miR-19b-3p + hsa-miR-196a-5p + hsa-miR-33b-3 p; (F)9 biomarker combinations. The abscissa is 100-Specificity (Specificity); the ordinate is Sensitivity (Sensitivity).

Detailed Description

Unless otherwise defined, technical terms used in the present application have the meanings commonly understood by those skilled in the art to which the present invention belongs.

The present application relates to a method useful for the early diagnosis of colorectal cancer, comprising the steps of: 1) collecting a biological sample; 2) Determining an exosome miRNA expression level of a biomarker of the biological sample, wherein the marker is one or more of hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and hsa-miR-33b-3 p; and 3) comparing the exosome miRNA expression level detected in step 2) with the corresponding biomarker expression level in the population to determine whether the subject has colorectal cancer. The miRNA biomarkers and the internal reference (hsa-miR-let-7d) have the following sequences.

As used herein, the term "subject" refers to an individual who has or is suspected of having a disease, and "subject" in predicting a susceptibility may also include healthy individuals. The term is often used interchangeably with "patient," "subject to be treated," and the like.

As used herein, "population" generally refers to a healthy population. When a particular analysis is performed for a disease, "population" may also refer to a person who does not have the disease but who has other diseases. For example, for colorectal cancer, the population may include healthy people or non-colorectal cancer patients, including adenomas, enteritis, polyps, and some other types of cancer patients. In addition, some individuals may be designated as "populations" based on characteristics such as age, smoking, alcohol abuse, and individual health. The "exosome miRNA levels" in a population can be determined by assaying a sufficient number of individuals.

In addition, the method can be used for predicting the colorectal cancer stage of the subject, and suggestions can be made on treatment of the subject according to the colorectal cancer stage, such as further colorectal endoscopy, surgery, medication guidance and no further action on the subject.

The step 2) of detecting the exosome miRNA comprises the steps of precipitating exosomes from a biological sample, extracting the exosome miRNA and measuring the expression level by using a marker specific RT-PCR primer and a fluorescent quantitative primer. The inventors designed corresponding primer pairs for the gene sequences of each biomarker, which also included primers required for hsa-miR-let-7d as an internal reference.

In some embodiments, the primers and corresponding probes corresponding to the biomarkers used in the method are available from commercial orders. For example, the exosome miRNA marker primers used in the examples herein include specific miRNA stem-loop RT-PCR primers purchased from synthetic production by bio-engineering (shanghai) gmbh.

In the embodiment of the method, the 5' end reporter fluorophore selected from the fluorescent probe can be FAM, JOE, TET, HEX, Cy3, Texas Red, Rox or Cy 5; the quenching group at the 3' end is BHQ1, BHQ2, BHQ3, TAMRA, DABCYL or MGB.

The biological sample is mainly from body fluid of a subject, including blood, serum, plasma, feces, milk, ascites, urine, tissue biopsy and the like, and preferably plasma, serum and feces.

In the method, the age, smoking and drinking conditions, the family history of diabetes mellitus and colorectal cancer and the like of the subject can be used as reference indexes, so that more accurate hierarchical risk prediction is provided for diagnosis of colorectal cancer.

In some embodiments, a diagnostic model based on exosome miRNA expression levels is constructed using statistical methods selected from the following: rank sum test, multiple linear regression, principal component analysis, decision trees, random forests, Probit regression, logistic regression, cluster analysis, neural networks, bayes and non-bayes methods, and the like. Can adopt x ²Tests, paired t-tests, and nonparametric rank-sum tests compare differences in miRNA expression levels among the different study groups. The diagnostic value of miRNA biomarkers (or combinations thereof) was confirmed by computational ROC curve analysis.

The method can provide reference information outside the existing colorectal cancer monitoring mode for a clinician, and can assist the clinician in evaluating and grading the risk of the patient suffering from colorectal cancer and planning the action to be taken subsequently.

The methods of the invention are useful for the prognosis of colorectal cancer. For example, biological samples are taken from the subject before and after treatment or during treatment, respectively, and exosome miRNA expression levels are detected as described above, and the efficacy of current treatment regimens can be determined by dynamic monitoring of miRNA expression levels.

The combination of the biomarkers miRNA provides a new method useful for determining the pathotype and stage of colorectal cancer lesions. In addition, the expression level of exosome mirnas in the biological sample has a correlation with the presence of pre-malignant or preclinical disorders in the patient. Therefore, the method can also be used for predicting the existence of colorectal cancer, benign and malignant colorectal cancer and colorectal cancer metastasis possibility.

The measurement method of the biomarker exosome miRNA is selected from one or more of the following methods: real-time fluorescence quantitative PCR, Northern blotting method, digital PCR, microarray chip method, high-throughput sequencing, nano-gold labeling method, high-resolution dissolution curve technology and time-of-flight mass spectrometry.

As used herein, "and/or" refers to any one or any combination of its preceding and following objects. For example, "A, B and/or C" may encompass A, B, C, "a and B," a and C, "" B and C, "and" A, B and C.

Disclosed herein are exosome miRNA markers associated with colorectal cancer auxiliary diagnosis and applications thereof. The marker is one or more of hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p and hsa-miR-33b-3 p. The exosome miRNA plays an important role in the processes of tumorigenesis, invasion, metastasis and the like, and researches show that the content wrapped by exosomes is directly from parental cells and has good stability and integrity, so that the exosome miRNA serving as a novel marker has the inherent advantages of strong diagnosis capability, high stability, easiness in acquisition and the like. The development of such markers will provide new diagnostic and therapeutic directions for a variety of diseases, including cancer. Disclosed herein are preferred combinations of exosome miRNA markers for colorectal cancer diagnosis, which combined subject operating characteristic (ROC) curve analysis has an area under the curve (AUC) of up to 0.964 with an accuracy of 94.19% and a specificity of 96.26%.

The invention is further described below by way of examples.

Example 1: extraction of plasma exosomes

A brief process for the extraction of exosomes from plasma is as follows:

1. the test plasma was removed and centrifuged at 3000 Xg for 15 minutes to remove some debris and some insoluble components.

2. The exosome extraction kit was an ExoQuick kit (EXOQ5TM-1, SBI). The supernatant was transferred to a new 1.5mL EP tube, to which an appropriate amount of ExoQuick-TC was added, and both were thoroughly shaken and mixed using a vortex shaker.

3. The plasma, well mixed with ExoQuick-TC, was incubated at 4 ℃ for more than 12 h.

4. The incubated ExoQuick-TC/plasma mixture was centrifuged at 1500 Xg for 30 min and the exosomes were precipitated as beige solids at the bottom of the EP tube.

5. The supernatant was discarded and the exosomes were resuspended in 1 x PBS solution for use.

Example 2: extraction of exosome miRNA

Blood plasma RNA extraction kit (AM1556) manufactured by ABI company is selected, and according to the instruction of the kit, 200 mu l of exosome weight suspension is sucked for each sample to extract RNA, and finally 100 mu l of DEPC water is used for dissolving.

Example 3: preparation of cDNA

miRNA reverse transcription reaction system

Reagents for formulating a reverse transcription reaction system were purchased from Biotechnology engineering (Shanghai) Ltd, and included M-MuLV reverse transcriptase (Cat: B600005), RNase inhibitor (Cat: B600008), dNTP Mix 10mM (Cat: B500056), and the reverse transcription reaction system was as follows:

TABLE 1 reverse transcription reaction System

Reverse transcription procedure:

30min at 16 ℃, 30min at 42 ℃, 5min at 85 ℃ and cooling at 4 ℃.

Example 4: real-time fluorescence PCR detection of miRNA expression level

In this example, real-time fluorescent quantitative PCR is taken as an example to detect the expression level of miRNA. The miRNA detection markers are hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p and hsa-miR-33b-3p, and the internal reference is hsa-let-7 d. Primers and probes were designed for these ten mirnas as follows:

Hsa-miR-320a-3p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCGCCC-3’(SEQ ID NO：10)

a forward primer: 5'-GCGAAAAGCTGGGTTGAGA-3' (SEQ ID NO: 19)

And (3) probe: NO 7:5 '-Cy 5-GGATACGACTCGCCC-BHQ 3-3' (SEQ ID NO: 29)

Hsa-miR-19a-3p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTT-3’(SEQ ID NO：11)

a forward primer: 5'-GCGTGTGCAAATCTATGCAA-3' (SEQ ID NO: 20)

And (3) probe: 5 '-Texas Red-CTGAGTCGTATCCAGTGCG-BHQ 2-3' (SEQ ID NO: 30)

Hsa-miR-19b-3p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTT-3’(SEQ ID NO：12)

a forward primer: 5'-CGTGTGCAAATCCATGCAA-3' (SEQ ID NO: 21)

And (3) probe: 5 '-HEX-ACTGAGTCGTATCCAGTGCG-BHQ 1-3' (SEQ ID NO: 31)

Hsa-miR-31-5p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGCTAT-3’(SEQ ID NO：13)

A forward primer: 5'-GCGAGGCAAGATGCTGGC-3' (SEQ ID NO: 22)

And (3) probe: 5 '-FAM-GCTGTCGTATCCAGTGCG-BHQ 2-3' (SEQ ID NO: 32)

Hsa-miR-92a-3p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGGC-3’(SEQ ID NO：14)

a forward primer: 5'-GCGTATTGCACTTGTCCCG-3' (SEQ ID NO: 23)

And (3) probe: 5 '-Texas Red-GATACGACACAGGCCG-BHQ 2-3' (SEQ ID NO: 33)

Hsa-miR-196a-5p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCCAAC-3’(SEQ ID NO：15)

a forward primer: 5'-CGCGCGTAGGTAGTTTCATGTT-3' (SEQ ID NO: 24)

And (3) probe: 5 '-CY 5-TGGGGTCGTATCCAGTGC-BHQ 1-3' (SEQ ID NO: 34)

Hsa-miR-409-5p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATGCAA-3’(SEQ ID NO：16)

a forward primer: 5'-GCGAGGTTACCCGAGCAACT-3' (SEQ ID NO: 25)

And (3) probe: 5 '-FAM-TGCATGTCGTATCCAGTGC-BHQ 2-3' (SEQ ID NO: 35)

Hsa-miR-520d-5p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGGCT-3’(SEQ ID NO：17)

a forward primer: 5'-CGCGCGCACAAAGGGA-3' (SEQ ID NO: 26)

And (3) probe: 5 '-HEX-CCCGTCGTATCCAGTGC-BHQ 2-3' (SEQ ID NO: 36)

Hsa-miR-33b-3p primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGCTG-3’(SEQ ID NO：18)

a forward primer: 5'-GCAGTGCCTCGGCAGTG-3' (SEQ ID NO: 27)

And (3) probe: 5 '-Texas Red-GCCCGTCGTATCCAGTG-BHQ 2-3' (SEQ ID NO: 37)

miRNA detection reference gene let-7 primer group

Reverse transcription primer:

5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT-3’(SEQ ID NO：38)

A forward primer: 5'-GCCCGCAGAGGTAGTAGGTTGC-3' (SEQ ID NO: 39)

And (3) probe: 5 '-FAM-CTGGATACGACAACTAT-BHQ 1-3' (SEQ ID NO: 40)

According to the design principle of miRNA stem-loop method primers, the miRNAs share the same reverse primer group:

5’-AGTGCAGGGTCCGAGGTATT-3’(SEQ ID NO：28)

and (3) detecting the expression level of miRNA, and carrying out 3 times of repeated PCR reactions on each sample, wherein the total volume of each reaction system is 20 mu l, and 10 mu l of PCR reaction solution, 5 mu l of primer mixture and 5 mu l of PCR template are included (see the following table 2).

TABLE 2 reaction procedure for detection of miRNA expression level

Example 5: determining the sensitivity and specificity of plasma in colorectal cancer patients, non-colorectal cancer populations (including benign patients, other types of cancer interfering and healthy populations)

Using 845 samples from patients pathologically assigned colorectal cancer ("case group") and 728 samples from patients assigned to non-colorectal cancer ("control group"), the control group comprised 224 enteritis patients, 232 patients pathologically assigned to non-cancerous colorectal polyps, 198 patients pathologically assigned to non-cancerous colorectal adenomas, 74 other types of samples (specifically, containing 27 colorectal healthy abnormal samples and 47 other cancers: 22 gastric cancer, 10 esophageal cancer, 15 liver cancers) (see table 3), all collected from beware medical testing center, beijing. Colorectal cancer samples of the case group included all stages and common typing of the disease. Colorectal cancer patients are diagnosed by colonoscopy, imaging and pathological diagnosis, the sample stage is based on the international TNM stage standard, and the sample typing is determined according to a tissue biopsy and immunohistochemical method. Control group samples included common types of benign disorders seen throughout the study population and some other types of cancer interfering samples and some colorectal healthy non-abnormal samples. A complete clinical pathology report was obtained after surgery, including patient age, smoking history, race, staging, typing and coding collection site for each sample.

TABLE 3 colorectal cancer staging and other characteristics of the sample subjects collected

The study samples are shown in table 3 for a total of 1573 samples, with the case group including 845 patients pathologically identified as colorectal cancer, including patients of common colorectal cancer typing and stages. The control group consisted of 728 samples of patients identified as non-colorectal cancer, the control group consisted of 224 enteritis patients, 232 patients with pathology identified as non-cancerous colorectal polyps, 198 patients with pathology identified as non-cancerous colorectal adenomas, and 74 other types of samples (specifically, 27 colorectal healthy non-abnormal samples and 47 other cancers: 22 gastric cancers, 10 esophageal cancers, 15 liver cancers). The sample type studied was plasma and the level of exosome miRNA expression was detected in the 1573 samples above using real-time fluorescent PCR assay. To help determine the ability of these biomarker genes to distinguish symptomatic similar cancers from benign colorectal disease, all samples were obtained from the same clinical population (patients undergoing surgery based on the presence of colorectal polyps). All samples were collected prior to any intervention and prior to the known disease state. The disease state is then determined by pathological examination of ex vivo tissues. Plasma was collected using a single sample collection protocol and compliance was monitored. This ensures sample quality and eliminates the possibility of any collection, processing, and biological bias in the sample collection. These samples show that the average patient age among individuals with colorectal cancer (58.43 years) is higher than those with benign conditions or no abnormalities in health (55.68 years) and has a tendency to increase with the staged progression of disease manifestations (table 3). Overall, the distribution of colorectal cancer typing is similar to that seen in all colorectal cancer cases in the human population, with a higher proportion of adenocarcinomas (70%) than other typed colorectal cancers. The non-colorectal cancer controls under investigation represent common benign colorectal diseases including enteritis, intestinal polyps, adenomas, etc., and also include interfering samples of some other types of cancer and some healthy non-abnormal samples.

To determine the diagnostic ability of the selected biomarkers for different stages (especially early) of colorectal cancer, the detection levels of 9 miRNA markers in benign samples as well as colorectal cancer samples of different stages were compared (fig. 1). For early colorectal cancer samples, hsa-miR-320a-3P, hsa-miR-19b-3P and hsa-miR-196a-5P have high discrimination (P value is less than 0.001); the hsa-miR-31-5P, hsa-miR-19a-3P and hsa-miR-33b-3P show a certain significant difference (the P value is less than 0.05) on early colorectal cancer and benign diseases, but the effect is slightly poor.

Furthermore, it was compared whether the above 9 miRNA levels had statistically significant differences between benign conditions and samples of various subtypes of colorectal cancer (fig. 2). For adenocarcinoma, hsa-miR-320a-3P, hsa-miR-19a-3P, hsa-miR-31-5P, hsa-miR-92a-3P, hsa-miR-196a-5P, hsa-miR-409-5P and hsa-miR-33b-3P all have high discrimination (P value <0.001), while hsa-miR-19b-3P and hsa-miR-520d-5P have slightly poor discrimination (P value <0.01) for adenocarcinoma. For mucinous adenocarcinomas, hsa-miR-320a-3P, hsa-miR-31-5P, hsa-miR-196a-5P and hsa-miR-19b-3P have very high degrees of discrimination (P value < 0.001). For signet ring cell carcinoma, hsa-miR-320a-3P, hsa-miR-92a-3P, hsa-miR-196a-5P and hsa-miR-33b-3P have high discrimination (P value <0.001), while hsa-miR-19b-3P and hsa-miR-409-5P have slightly lower discrimination (P value <0.05) for signet ring cell carcinoma. For other types of colorectal cancer, other miRNA markers, except hsa-miR-92a-3P, showed better discrimination (P-value < 0.01).

Detection of single miRNA levels is superior to detection of multiple miRNA marker levels in terms of simple handling and cost reduction. However, a single miRNA level may not provide information on the inherent diversity of complex diseases, and therefore it is often necessary to construct diagnostic models of multiple markers. The multiple marker diagnosis models need to be performed by using a statistical analysis method, and a multiple miRNA marker diagnosis model is constructed by taking a logistic regression model as an example.

The logistic regression model is constructed in the following way: samples were divided into case and control groups and then regression coefficients were optimized using commercially available software packages (IBM SPSS Statistics 24 and MedCalc11.4.2.0, available from IBM and MedCalc, respectively). There is a regression coefficient for each marker, plus a deviation parameter, to maximize the likelihood that the logistic regression model will be used to train the data. After training, the set of regression coefficients defines a logistic regression model. One skilled in the art can readily use this diagnostic model to predict the likelihood of any new sample being a case or control by substituting the expression levels of miRNA markers into a logistic regression equation.

The inventors compared the efficacy of single and multiple marker combinations for diagnosing colorectal cancer, respectively, by constructing logistic regression models and analyzing the results to obtain a better combination. Wherein, the three markers hsa-miR-320a-3p, hsa-miR-19B-3p and hsa-miR-196a-5p have excellent diagnosis performance on colorectal cancer when being used independently (see figures 3A, 3B and 3C). When the diagnosis effectiveness of the multi-marker combination is analyzed, hsa-miR-320a-3p + hsa-miR-31-5p + hsa-miR-409-5p and hsa-miR-19b-3p + hsa-miR-196a-5p + hsa-miR-33b-3p can provide more excellent colorectal cancer diagnosis performance (see figures 3D and 3E). In order to obtain a better colorectal cancer diagnosis effect, the expression level data of 9 miRNAs are comprehensively analyzed, and the result shows that the 9 miRNA markers can obtain a better diagnosis effect by combined analysis, and the AUC value can reach 0.964 (see figure 3F). The information related to the area under the curve (AUC) of the Receiver Operating Characteristic (ROC) curve analysis obtained for each marker and combination is summarized in table 4 below.

TABLE 4 Area Under Curve (AUC) of selected markers and Combined Receiver Operating Characteristics (ROC) Curve analysis

In order to determine the sensitivity of the identification of the selected markers and combinations for different stages of colorectal cancer, in particular early stages (stage I or II), the diagnostic sensitivity of colorectal cancer samples for each stage, in particular early stages (stage I or II) where marker detection is most important, is determined and compared. The results show that the diagnosis sensitivity of a single marker to early colorectal cancer is slightly lower than the combined effect of the markers, the diagnosis sensitivity of each marker combination is increased along with the increase of the stage of colorectal cancer, the combined use of the hsa-miR-320a-3p + hsa-miR-31-5p + hsa-miR-409-5p and 9 markers can diagnose that the sensitivity of the stage I and the stage II reaches over 90 percent, and the early colorectal cancer diagnosis method shows better diagnosis capability (see table 5).

TABLE 5 sensitivity of each marker and combination in different stages of colorectal cancer

To assess the sensitivity of the selected markers and combinations for the identification of different types of colorectal cancer, particularly the higher-ranking adenocarcinomas, the diagnostic sensitivity of the markers and combinations in each common type of colorectal cancer was compared. The results show that the sensitivity of hsa-miR-320a-3p, hsa-miR-320a-3p + hsa-miR-31-5p + hsa-miR-409-5p to the colorectal cancer in various types can be higher than 95%, the sensitivity of hsa-miR-196a-5p in combination with 9 markers to the colorectal cancer in various types is more than or equal to 90%, and the diagnosis effect is very excellent (see Table 6).

TABLE 6 sensitivity of each marker and combination in different colorectal cancer typing

To evaluate the specificity of the two marker combinations for the identification of a control group of non-colorectal cancer populations, study samples including colorectal common diseases such as enteritis, polyps, adenomas, etc., and also including some other control samples (other types of cancer interfering samples and some healthy non-abnormal samples), the specificity of the two combinations in non-colorectal cancer, especially in the disease or other cancer samples that are easily confused with colorectal cancer, was compared. The result shows that the selected miRNA marker has good distinguishing capacity on common interference samples of colorectal cancer when being used alone, and the specificity on colorectal polyps and adenomas is over 96 percent when the hsa-miR-320a-3p + hsa-miR-31-5p + hsa-miR-409-5p and the hsa-miR-19b-3p + hsa-miR-196a-5p + hsa-miR-33b-3p are used in combination, and the joint analysis of the 9 markers can improve the capacity of distinguishing the interference samples to 97.8 percent again. (see Table 7).

TABLE 7 specificity of each marker and combination in control samples

To evaluate the sensitivity of the selected markers and combinations for the identification of different differentiation levels (see for details the UICC/AJCC TNM staging system) for colorectal cancer, the sensitivity of the two combinations in each differentiation level (in particular low and undifferentiated samples) was compared. The result shows that the diagnosis effect of the single miRNA as the marker on the colorectal cancer with different differentiation levels is slightly poor, the diagnosis specificity of the hsa-miR-320a-3p + hsa-miR-31-5p + hsa-miR-409-5p and the hsa-miR-19b-3p + hsa-miR-196a-5p + hsa-miR-33b-3p can reach more than 92% when the single miRNA is used in combination, the comprehensive diagnosis specificity of the 9 markers for the low-differentiation colorectal cancer can be improved to 96% when the single miRNA is used in combination, and the sensitivity tends to increase along with the increase of the differentiation degree of the colorectal cancer (see Table 8).

TABLE 8 sensitivity of markers and combinations in different colorectal cancer differentiation levels

According to the technical scheme provided by the invention, the sensitivity and specificity of colorectal cancer detection are improved by jointly detecting multiple exosome miRNAs, so that the correctness and reliability of the detection result are ensured.

The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; one of ordinary skill in the art will appreciate that: modifications can be made to the technical solutions described in the foregoing embodiments, or some or all of the technical features can be equivalently replaced; the modifications or the substitutions do not cause the essence of the corresponding technical solutions to depart from the scope of the technical solutions of the embodiments, and the corresponding technical solutions are all covered in the scope of the description.

SEQUENCE LISTING

<110> Beijing Aikelen medical science and technology Co., Ltd

<120> detection kit and method for identifying colorectal cancer status by exosome miRNA marker

<130> 21800CI

<160> 41

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Claims

1. A test kit for use in the diagnosis of a colorectal cancer state in a subject, comprising a detection reagent for detecting the level of a miRNA biomarker selected from the group consisting of hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and hsa-miR-33b-3p, and any combination thereof, in a biological sample from the subject.

2. The test kit of claim 1, wherein the miRNA biomarker comprises hsa-miR-320a-3p, hsa-miR-19b-3p, or hsa-miR-196a-5 p; comprises hsa-miR-320a-3p, hsa-miR-31-5p and hsa-miR-409-5 p; or comprises hsa-miR-19b-3p, hsa-miR-196a-5p and hsa-miR-33b-3 p.

3. The detection kit of claim 1 or 2, wherein the miRNA biomarkers are hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and hsa-miR-33b-3 p.

4. The test kit of any of claims 1-3, wherein the colorectal cancer status comprises colorectal cancer susceptibility or presence, progression, subtype, staging and/or differentiation status of colorectal cancer.

5. The test kit of any of claims 1-4, wherein the colorectal cancer status is stage I or stage II colorectal cancer, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19b-3p and/or hsa-miR-196a-5 p; the colorectal cancer state is adenocarcinoma, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19a-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p and/or hsa-miR-33b-3 p; the colorectal cancer state is mucus adenocarcinoma, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-31-5p, hsa-miR-196a-5p and/or hsa-miR-19b-3 p; the colorectal cancer state is signet ring cell carcinoma, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-92a-3p, hsa-miR-196a-5p and/or hsa-miR-33b-3 p; or the colorectal cancer state is other types of colorectal cancer, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p and/or hsa-miR-33b-3 p.

6. The detection kit of any one of claims 1-5, wherein the detection reagents for detecting miRNA biomarker levels comprise reverse transcription primers, PCR amplification primer pairs, and/or Taqman probes.

7. The test kit according to any one of claims 1 to 6, wherein:

the detection reagent for detecting the level of hsa-miR-320a-3p comprises: comprises the amino acid sequence of SEQ ID NO: 10, and a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 19 and 28; and/or comprises SEQ ID NO: 29, and a Taqman probe;

the detection reagent for detecting the level of hsa-miR-19a-3p comprises: comprises the amino acid sequence of SEQ ID NO: 11, and a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 20 and 28; and/or comprises SEQ ID NO: 30, a Taqman probe;

the detection reagent for detecting the level of hsa-miR-19b-3p comprises: comprises the amino acid sequence of SEQ ID NO: 12; comprising SEQ ID NO: 21 and 28; and/or comprises SEQ ID NO: 31, a Taqman probe;

the detection reagent for detecting the level of hsa-miR-31-5p comprises: comprises the amino acid sequence of SEQ ID NO: 13, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 22 and 28; and/or comprises SEQ ID NO: 32, a Taqman probe;

The detection reagent for detecting the level of hsa-miR-92a-3p comprises: comprising SEQ ID NO: 14, and a reverse transcription primer; comprising SEQ ID NO: 23 and 28; and/or comprises SEQ ID NO: 33, a Taqman probe;

the detection reagent for detecting the level of hsa-miR-196a-5p comprises: comprising SEQ ID NO: 15, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 24 and 28; and/or comprises SEQ ID NO: 34, 34 is a Taqman probe;

the detection reagent for detecting the level of hsa-miR-409-5p comprises: comprises the amino acid sequence of SEQ ID NO: 16; comprises the amino acid sequence of SEQ ID NO: 25 and 28; and/or comprises SEQ ID NO: 35, a Taqman probe with a sequence shown in the specification;

the detection reagent for detecting the level of hsa-miR-520d-5p comprises: comprises the amino acid sequence of SEQ ID NO: 17; comprises the amino acid sequence of SEQ ID NO: 26 and 28; and/or comprises SEQ ID NO: 36, a Taqman probe; and

the detection reagent for detecting the level of hsa-miR-33b-3p comprises: comprises the amino acid sequence of SEQ ID NO: 18, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 27 and 28; and/or comprises SEQ ID NO: 37, and a Taqman probe having the sequence shown in the specification.

8. The test kit according to any one of claims 1 to 7, further comprising a detection reagent for detecting the level of hsa-miR-let-7d as an internal reference gene; the detection reagent comprises: comprises the amino acid sequence of SEQ ID NO: 38; comprises the amino acid sequence of SEQ ID NO: 39 and 28; and/or comprises SEQ ID NO: 40 in sequence shown in the specification.

9. The test kit of any one of claims 1-8, further comprising reagents for isolating exosomes from the biological sample; optionally, further comprising reagents for extracting miRNA from the exosomes.

10. The test kit of any one of claims 1-9, further comprising instructions describing a determination of colorectal cancer status in the subject based on logistic regression from the miRNA biomarker levels.

11. The test kit of any one of claims 1-10, wherein the biological sample is selected from the group consisting of blood, serum, plasma, stool, milk, ascites, urine, and tissue biopsy; preferably plasma, serum or feces.

12. A method of identifying colorectal cancer status in a subject, comprising:

1) isolating exosomes from a biological sample from the subject;

2) Detecting a level of a miRNA biomarker in the exosomes, the miRNA biomarker selected from the group consisting of hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p and hsa-miR-33b-3p and any combination thereof; and

3) comparing the miRNA biomarker levels detected in step 2) with the levels of corresponding mirnas in a population to determine the colorectal cancer status in the subject.

13. The method of claim 12, wherein the miRNA biomarkers comprise a miRNA biomarker comprising hsa-miR-320a-3p, hsa-miR-19b-3p, or hsa-miR-196a-5 p; comprises hsa-miR-320a-3p, hsa-miR-31-5p and hsa-miR-409-5 p; or comprises hsa-miR-19b-3p, hsa-miR-196a-5p and hsa-miR-33b-3 p.

14. The method of claim 12 or 13, wherein the miRNA biomarkers are hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p, and hsa-miR-33b-3 p.

15. The method of any one of claims 12-14, wherein the colorectal cancer status comprises colorectal cancer susceptibility or presence, progression, subtype, stage and/or differentiation status of colorectal cancer.

16. The method of any one of claims 12-15, wherein the colorectal cancer status is colorectal cancer stage I or II, the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19b-3p, and/or hsa-miR-196a-5 p; the colorectal cancer state is adenocarcinoma, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19a-3p, hsa-miR-31-5p, hsa-miR-92a-3p, hsa-miR-196a-5p, hsa-miR-409-5p and/or hsa-miR-33b-3 p; the colorectal cancer state is mucus adenocarcinoma, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-31-5p, hsa-miR-196a-5p and/or hsa-miR-19b-3 p; the colorectal cancer state is signet ring cell carcinoma, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-92a-3p, hsa-miR-196a-5p and/or hsa-miR-33b-3 p; or the colorectal cancer state is other types of colorectal cancer, and the miRNA biomarker is hsa-miR-320a-3p, hsa-miR-19b-3p, hsa-miR-31-5p, hsa-miR-196a-5p, hsa-miR-409-5p, hsa-miR-520d-5p and/or hsa-miR-33b-3 p.

17. The method of any one of claims 12-16, wherein the detection of the miRNA biomarker levels in step 2) comprises using miRNA biomarker-specific detection reagents comprising reverse transcription primers, PCR amplification primer pairs, and/or Taqman probes.

18. The method of any one of claims 12-17, wherein:

the detection reagent for detecting the level of hsa-miR-19b-3p comprises: comprises the amino acid sequence of SEQ ID NO: 12; comprises the amino acid sequence of SEQ ID NO: 21 and 28; and/or comprises SEQ ID NO: 31, a Taqman probe;

the detection reagent for detecting the level of hsa-miR-92a-3p comprises: comprises the amino acid sequence of SEQ ID NO: 14, a reverse transcription primer; comprises the amino acid sequence of SEQ ID NO: 23 and 28; and/or comprises SEQ ID NO: 33, a Taqman probe;

The detection reagent for detecting the level of hsa-miR-196a-5p comprises: comprising SEQ ID NO: 15, and a reverse transcription primer; comprising SEQ ID NO: 24 and 28; and/or comprises SEQ ID NO: 34, 34 is a Taqman probe;

the detection reagent for detecting the level of hsa-miR-409-5p comprises: comprising SEQ ID NO: 16; comprises the amino acid sequence of SEQ ID NO: 25 and 28; and/or comprises SEQ ID NO: 35, a Taqman probe with a sequence shown in the specification;

19. The method of any one of claims 12-18, wherein step 2) further comprises detecting the level of hsa-miR-let-7d as an internal reference gene; the detection reagent for detecting the level of hsa-miR-let-7d comprises: comprises the amino acid sequence of SEQ ID NO: 38; comprises the amino acid sequence of SEQ ID NO: 39 and 28; and/or comprises SEQ ID NO: 40 in sequence shown in the specification.

20. The method of any one of claims 12-19, wherein step 3) comprises determining colorectal cancer status in the subject based on logistic regression from the miRNA biomarker levels.

21. The method of any one of claims 12-20, wherein the biological sample is selected from the group consisting of blood, serum, plasma, stool, milk, ascites, urine, and tissue biopsy; preferably plasma, serum or faeces.